Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

doi: 10.1038/labinvest.2015.149

Figure Lengend Snippet: Figure 3 Notch signaling pathway was activated in CLF. (a) Notch-1, 2, 3, 4 mRNA. (b) JAG1, 2, and DLL1, 3, 4 mRNA. (c) Hes1, Numb, and RBPJк mRNA. All mRNA were quantified with RT-PCR and normalized to GAPDH mRNA (n = 6 per group). (d) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb protein expression were quantified via immunoblotting and normalized to GAPDH (n = 6 per group), and (e) densitometric analysis of protein bands. *Po0.05, **Po0.01. Sham, Sham group; 1 wM, BDL-1w group; 2 wM, BDL-2w group; 3 wM, BDL-3w group; 4 wM, BDL-4w group.

Article Snippet: Rabbit polyclonal antibodies to Notch4 (sc-5594), JAG1 (sc-8303), DLL1 (sc-9102), mouse monoclonal antibody to Sox9 (E-9, sc-166505), and goat polyclonal antibody to JAG2 (sc-34475) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

doi: 10.1038/labinvest.2015.149

Figure Lengend Snippet: Figure 6 Inhibition of the Notch signaling pathway reduced the progression of liver fibrosis. (a) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb, protein bands on the left (immunoblot), and the histogram depicts the densitometric analysis of protein bands (n = 6 per group). (b) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb mRNA were measured via RT-PCR and normalized to GAPDH mRNA (n = 6 per group). (c) Sirius Red staining (×100). (d) α-SMA immunostaining (×200). (e) Hydroxyproline content. (f) α-SMA, TNF-α, TGF-β1, MCP-1, Col(1), and Col (4) mRNA were measured by RT-PCR and normalized to GAPDH mRNA (n = 6 per group). *Po0.05, **Po0.01. BDL, single bile duct ligation group; DAPT, BDL plus DAPT group; sham, sham group.

Article Snippet: Rabbit polyclonal antibodies to Notch4 (sc-5594), JAG1 (sc-8303), DLL1 (sc-9102), mouse monoclonal antibody to Sox9 (E-9, sc-166505), and goat polyclonal antibody to JAG2 (sc-34475) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Immunostaining, Ligation

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Abnormal expression of NOTCH1–4. (A) Full landscape of NOTCH1–4 expression profiles in multiple cancer types in the TIMER database. (B) TCGA-based UALCAN database screening and analysis showing a significant difference in the NOTCH1 and NOTCH4 expressions between normal tissue and PCa tissue (P=1.047e-6, 1.05e-1, 5.70e-2, 5.93e-5, respectively). (C) IHC staining of NOTCH1–4 in PCa or BPH tissues respectively [N (tumor) =15, N (BPH) =6]. (D) qRT-PCR results for the mRNA levels of PCa tissues and adjacent normal prostate tissues. (T =24, N =24). *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001. ns, no significant. PRAD, prostate adenocarcinoma. qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; BPH, benign prostate hyperplasia; IHC, immunohistochemistry; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Correlation between NOTCH1–4 expression and Gleason score or prognosis in PCa patients. (A) UALCAN database analysis showing that the transcriptional level of NOTCH3 and NOTCH4 is significantly enhanced with increasing Gleason score. (B) The Mantel–Cox test with GEPIA2 showing that the expression of NOTCH3 and NOTCH4 exhibited a positive correlation with prognosis in PCa patients. (C) Cox regression analysis showing that patients in the low NOTCH1, 3 and 4 groups had a significant better disease-free survival (DFS) than those in the high group (P=4.8e-2, 5.6e-3, and 2.0e-2, respectively). (D) Cox regression analysis showing that there was no significant difference in overall survival (OS) between high and low NOTCH family genes’ expression. *, P<0.05, **, P<0.01, ***, P<0.001 and ****, P<0.0001. GS, Gleason score; PRAD, prostate adenocarcinoma; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Neighbor gene network and interaction analyses of NOTCH1–4 in PCa patients. (A) Correlation heat map of NOTCH family genes and NOTCH-related genes in PCa patients. (B,C) Protein-protein interaction networks of differently expressed NOTCH family genes. PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques:

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Characterization of genetic alterations and mutations of NOTCH1–4. (A) Mutational frequencies of NOTCH1, NOTCH2, NOTCH3 and NOTCH4 in PCa were 7%, 8%, 5%, and 5%, respectively. (B) Top 10 most significant mutation-related genes of NOTCH1–4. (C) Wayne chart showing the shared mutation-related genes of NOTCH1–4. (D) Bar graph showing the top 20 results from the GO enrichment analysis. (E) GO enrichment analysis showing the gene networks. GO, gene ontology; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Common differentially expressed genes among NOTCH1–4 mutations

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Derivative Assay

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: NOTCH1–4 mutations and drug sensitivity. (A) Volcano plots showing that multiple cancer cell types with the NOTCH1 mutation were sensitively targeted by BMS-754807, linsitinib, saracatinib and erlotinib (left-hand panel). Tumor cells with the NOTCH2 mutation showed potential antagonism against entospletinib (right-hand panel). (B) Scatter plots showing that tumor cells were significantly suppressed by the use of BMS-754807, linsitinib, saracatinib, erlotinib and entospletinib in the NOTCH1 mutation group compared with the wild-type group (P<0.05 for all). *, P<0.05, **, P<0.01, and ***, P<0.001. IC50, half maximal inhibitory concentration; GDSC, genomics of drug sensitivity in cancer; Mut, mutation.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis, Concentration Assay

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: NOTCH mutation-related drug-sensitivity analyzed using ANOVA

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Correlation between differently expressed NOTCH1–4 and lymphatic metastasis or immune cell infiltration. (A) TIMER database analysis showing that patients with N1 stage had significant higher expression of NOTCH3 than those with N0 stage (P<0.05, N0 = no lymphatic metastasis, N1 = lymphatic metastasis). (B) Associations between NOTCH1–4 and lymphocytes infiltration in PCa patients. *, P<0.05, ***, P<0.001 and ****, P<0.0001. ns, no significant. PCa, prostate cancer; PRAD, prostate adenocarcinoma; TIMER, tumor immune estimation resource.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 3. Decreased glutathione pools by CHAC1 reduces protein-SSG and aggravates APAP-induced hepatotoxicity and ferroptosis in APAP-injured mice liver. (A) Quantification of GSH in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (B) Quantification of GSSG in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad- CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (C) Western blot analysis of S-glu tathionylated proteins in the liver tissues of Chac1+/+ Ad-GFP and Chac1−/−Ad-GFP mice treated with 300 mg/kg APAP for 2 and 6 h. GAPDH was used as an internal reference, followed by quantification of relative levels of S-glutathionylated proteins after 6 h of APAP treatment (Data are mean ± SEM of n = 3 mice/ group, t-test). (D) Western blot analysis of S-glutathionylated proteins and CHAC1-FLAG protein in the liver tissues of Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. GAPDH was used as an internal reference. Statistical chart shows the relative expression levels of S-glutathionylated proteins 6 h after APAP treatment (Data are mean ± SEM of n = 4 mice/group, t-test). (E) Serum levels of ALT and AST in Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h (Data are mean ± SEM of n = 5 mice/group, t-test). (F) H&E staining, 4-HNE protein adduct staining in liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. Scale bars = 200 μm. The area of liver injury and immunohistochemical score for 4-HNE protein adduct staining were quantified (Data are mean ± SEM of n = 5 mice/group, t-test). APAP, acetaminophen; GSH, glutathione; GSSG, oxidized glutathione; ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, haematoxylin and eosin; Chac1−/−, Chac1- deficient; Chac1+/+, wild-type controls.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Saline, Western Blot, Expressing, Staining, Immunohistochemical staining

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 4. Redox proteomic analysis reveals that the S-glutathionylation of Cys90 on ARF6 is regulated by CHAC1 in ferroptotic PMHs induced by APAP. (A) Flowchart outlining the key experimental procedures for proteomic analysis of S-glutathionylation. (B) Scatter plot illustrating the distribution of differential modification sites, sorted by the ratio of Ad-GFP + APAP/Ad-GFP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences, and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (C) Scatter plot showing the distribution of differential modification sites, sorted by the ratio of Ad-CHAC1 + APAP/Ad-GFP + APAP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (D) Venn diagram showing differentially modified sites under both APAP stimulation and CHAC1 overexpression (Fold change ≥1.2). (E) The heat map illustrating the union of differential modification sites in Ad-GFP, Ad-GFP + APAP, Ad-CHAC1, and Ad-CHAC1 + APAP comparison groups (CV < 0.1, fold change ≥1.2). (F) Scatter plot showing differentially modified sites under both APAP stimulation and CHAC1 overexpression; the order was sorted by the ratio of Ad-GFP + APAP/Ad-GFP (CV < 0.1, fold change ≥1.2). (G) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in PMHs. The secondary mass spectrum shows fragment ion information of the ARF6 C90 peptide segment. (H) Histogram showing the relative modification abundance of ARF6 C90 in different treatment groups, with glutathionylated peptides identified and quantified by LC-MS/MS (All values were normalized by the mean of the AdGFP-CON group, data are mean ± SEM of n = 2 biologically independent samples). (I) IP assay showing the expression of S-glutathionylated ARF6 in 293T cells overexpressing Myc-tagged ARF6. Whole cell lysates were used to confirm the expression of ARF6. (J) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in 293T cells overexpressing Myc-tagged ARF6. The secondary mass spectrum shows fragment ion infor mation of the ARF6 C90 peptide segment. PMH, primary mouse hepatocyte; IP, immunoprecipitation.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Modification, Over Expression, Comparison, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Immunoprecipitation